Brain serotonin levels were increased in immature chickens by ip injection of pargyline (75 mg/kg) and clorgyline (5 mg/kg) and by L-tryptophan (100 mg/kg) and imipramine (10 mg/kg) treatment. These treatments increased the circulating prolactin level and reduced the concentration of plasma growth hormone (GH). Treatment with para-chlorophenylalanine (PCPA, 100 mg/kg) reduced the brain serotonin content and the level of plasma prolactin. Treatment with these drugs in vivo similarly affected the basal level of prolactin release from pituitary glands in vitro, although it did not affect the basal level of GH release. The in vitro responsiveness of the pituitary gland to hypothalamic stimuli eliciting prolactin secretion was increased by in vivo pargyline and combined tryptophan: imipramine treatment but reduced by PCPA administration. The in vitro GH response to hypothalamic stimulation was reduced after the in vivo injection of pargyline, clorgyline and tryptophan: imipramine. The hypothalami from clorgyline and tryptophan: imipramine treated birds induced a greater stimulation of in vitro prolactin secretion from control pituitary glands than hypothalami from controls birds, whereas the GH releasing activity was reduced. These results suggest that serotonin stimulates prolactin secretion in chickens by increasing pituitary responsiveness to hypothalamic releasing factors and by increasing the prolactin releasing activity of the hypothalamus. Serotonin appears to suppress GH secretion by reducing pituitary sensitivity to releasing factors and by reducing hypothalamic GH releasing activity.